Differentiation of Common Respiratory Viruses and SARS

描述(申请人提供)：2002/2003年的SARS疫情证明了新出现的传染病病原体的致命性和传染性。SARS的临床表现与其他呼吸道感染相似，使诊断复杂化，但分子诊断方法将SARS与危险性较小的呼吸道感染区分开来。根据与美国国防部的合同，爱达荷州技术公司开发了剃须刀，这是一种电池操作的手持式聚合酶链式反应仪器，利用荧光检测特定的扩增产物，并使用软件自动判断样本为阳性或阴性。这项申请提出了四个具体目标，涉及将军用剃刀转变为民用探路者，最初的应用是区分常见的呼吸道病毒和SARS。具体目标I涉及对现有仪器进行工程改造，以实现高分辨率的热熔化曲线分析。这包括对光学系统的改变，以便可以使用新型dsDNA结合染料LC Green进行检测，以及改变加热器以允许精细的温度控制。特定目标2修改了爱达荷州技术公司现有的手动DNA纯化方案，以从人类鼻咽吸液样本(呼吸道感染诊断的首选矩阵)中提纯RNA。特定目标3高分辨率热熔化曲线的多重分析要求单个扩增产物的熔融特性是唯一的。针对每一种RNA呼吸道病毒：RSV、流感A和B以及SARS冠状病毒开发了一套特异的扩增产物，每种病毒都具有分离良好、易于区分的熔融图谱。产生这些扩增产物的一组引物在多重到4级所需的组合中进行测试。用于执行反转录/聚合酶链式反应的冻干试剂被开发出来，并且是平台的组成部分。具体目标4建议将探路者集成到一个工作站中，自动化核酸制备并无缝地准备PCR反应。考虑了几种可能的方法，用于细胞裂解、核酸提取和低功率格式的纯化。

DESCRIPTION (provided by applicant): The SARS outbreak of 2002/2003 demonstrated the lethality and communicability of emerging infectious agents. Clinical presentation of SARS is similar to other respiratory infections, complicating diagnosis, however diagnosis by molecular means differentiates SARS from less dangerous respiratory infections. Under contract to the US Department of Defense, Idaho Technology developed the RAZOR, a battery-operated hand-portable PCR instrument utilizing fluorescence to detect -specific amplification products and software to automatically call samples as either positive or negative. This application proposes four specific aims involving the transforming the military RAZOR to the civilian PathFinder with an initial application to differentiate common respiratory viruses from SARS. Specific Aim I involves engineering changes to the existing instrument to allow high-resolution thermal melting curve analysis. This includes changes to the optical system such that detection using the novel dsDNA binding dye LCGreen is possible and changes to the heaters to allow fine temperature control. Specific Aim 2 modifies Idaho Technology's existing manual DNA purification protocol to purify RNA from human nasopharangeal aspirate samples (the matrix of choice for diagnosis of respiratory infections). Specific Aim 3 Multiplex analysis of high-resolution thermal melting curves requires that the melting characteristic of individual amplification products be unique. A set of amplicons specific for each of the RNA respiratory viruses: RSV, influenza A and B and the SARS coronavirus are developed with each have well-separated, readily differentiated melting profiles. Sets of primers to generate these amplification products are tested in combinations necessary for multiplexing to a level of 4. Lyophilized reagents for performing the reverse transcription/PCR reactions are developed and are an integral part of platform. Specific Aim 4 proposes to integrate PathFinder into a workstation automating nucleic acid preparation and seamlessly preparing the PCR reaction. Several possible methodologies for cell lysis, nucleic acid extraction and purification in a low power format are considered.